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Manipulating macrophages: increasing the effectiveness in anti-cancer therapy

Braster, R.

2017

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citation for published version (APA)Braster, R. (2017). Manipulating macrophages: increasing the effectiveness in anti-cancer therapy.

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1Rens Braster1, Tom O’Toole1, Marjolein van Egmond1,2

Methods 2014

Myeloid cells as effector cells for monoclonal antibody therapy of cancer

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AbstractMonoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.

1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT

Amsterdam, The Netherlands;2 Department of Surgery, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

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11. IntroductionMonoclonal antibodies (mAb) are increasingly used as therapeutic agents in autoimmune diseases, chronic inflammation and cancer. They can be used to deliver therapeutic drugs (including toxins or chemokines/cytokines) or as diagnostic tool (e.g. radioisotopes)1,2. However, mAbs additionally induce multiple cytotoxic effects. For instance, they can directly inhibit tumor growth by apoptosis induction, neutralizing growth stimulatory molecules like vascular endothelial growth factor (VEGF) or by blocking specific growth factor receptors (such as epithelial growth factor receptor; EGFR) (Figure 1A). It has been shown that direct effects can play a prominent role in clinical successes of mAb therapy. Anti-EGFR mAbs are for example used to treat patients with head and neck cancer or colorectal cancer, but are only effective in patients that express wild type KRAS, whereas mutations in the EGFR signalling pathway seriously interfere with therapeutic success3. Tumor-targeting mAbs can furthermore induce indirect cytotoxicity by activating components of the immune system. Most clinically used mAbs are of the IgG subclass, which have the potential to bind C1q (Figure 1B).

FIGURE 1. Schematic and simplified model of mAb-induced effector mechanisms.(A) Direct effects induced by mAbs include blocking of growth factor receptors, or induction of apoptosis. (B) mAbs can initiate complement-dependent cytotoxicity (CDC) after binding of C1q to its Fc domain, which will initiate the classical complement pathway. (C) Opsonization of tumor cells with mAbs results in killing by immune effectors cells via different mechanisms. Of note: it is not clear yet via which mechanisms neutrophils kill tumor cells, but they likely differ from NK cell-induced classical antibody-dependent cellular cytotoxiciy (ADCC).

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This will activate the complement cascade through the classical pathway, thereby opsonizing targets by deposition of C3b, which can lead to tumor lysis through the lytic pathway (complement-dependent cytotoxicity; CDC). The role of CDC in patients is not yet completely clear. It has however been shown that polymorphisms in the C1QA gene correlated with clinical responses in patients with follicular lymphoma after rituximab treatment, supporting the importance of CDC4. Both direct effects of mAbs and CDC have recently been reviewed elsewhere1,2,5.The Fc domain of IgG mAbs interacts with IgG Fc receptors (Fcγ receptors) (Figure 1C). As such, mAbs form a bridge between tumor cells and Fcγ receptor-expressing immune effector cells, which can lead to tumor cell death that is generally referred to as antibody dependent cellular cytotoxicity (ADCC). Four IgG subclasses have been defined in humans (IgG1, IgG2, IgG3, IgG4). Additionally, the human IgG receptor family includes several members (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb), which can be activating or inhibitory and which differ in their affinities for the IgG subclasses (Table 1). Characteristics of human and mouse Fc receptors have recently been extensively reviewed elsewhere6-8. It has been demonstrated that Fcg receptor-mediated mechanisms are involved in therapeutic efficacy in vivo for most mAbs. For instance, anti-gp75 mAb therapy did not prevent melanoma development in FcR γ-chain deficient mice that lack the activating FcγRI, FcγRIII and FcγRIV9. Moreover, mAb therapeutic efficacy was absent in mice lacking FcγRI, or after blocking FcγRIV (or both), whereas murine FcγRIII was not involved10-12. By contract, anti-tumor mAbs were more effective in preventing tumor development in mice that were deficient for the inhibitory receptor FcγRII13. Fc receptor polymorphisms that affect affinity for IgG (FcγRIIa-131H/R and FcγRIIIa-158V/F) have been associated with clinical success of rituximab (anti-CD20), cetuximab (anti-EGFR) or trastuzumab (anti-HER-2; human epidermal growth factor receptor 2) therapy in lymphoma, colorectal or breast cancer, respectively14-16, demonstrating that Fcg receptor-mediated effector functions play an important role in cancer patients. It is not completely understood which Fcγ receptor-bearing cells provide cytotoxic activity in vivo. Traditionally, natural killer (NK) cells have been considered as main effector cells for ADCC17. NK cells are very effective killers of virus infected cells and tumor cells that lack MHC-I (Major Histocompatibility Complex-I)18. Furthermore, NK cells express FcγRIIIa, and the association between Fc receptor alleles (FcγRIIIa- V158allotype versus FcγRIIIa- F158 allotype), and clinical success of mAb therapy has been attributed to NK cells19. However, other cell populations can act as potential effector cells for mAb-mediated tumor regression, including the myeloid effector cells monocytes/macrophages and neutrophils, which will be the focus of this manuscript.

2. Macrophages as effector cells in mAb therapy of cancer.Macrophages are derived from circulating monocytes, and are potent regulators of the immune system in tissues during infections and wound healing20,21. Furthermore, it has been demonstrated that many tumors contain a considerable macrophage population, which are referred to as tumor-associated macrophages (TAMs). Macrophage presence has been correlated with tumor development. For instance, when invasive breast carcinomas were stained for CD68 (macrophage marker), an association between macrophage infiltration and poor prognosis of patients was observed22. By contrast, patients with colorectal cancer had a better prognosis when colon carcinomas were densely infiltrated with macrophages23,24. Thus, depending on the type of malignancy high macrophage numbers in the tumor is associated with

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either good or bad prognosis of patients22,25. It was furthermore recently demonstrated that TAMs, which were isolated from mouse breast carcinomas promoted tumor cell invasion in in vitro 3D assays, and as such have a tumor promoting function26. Nonetheless, these TAMs expressed Fcγ receptors and were capable of phagocytosing breast carcinoma cells in the presence of anti-CD142 mAbs. Furthermore, when macrophages were depleted with clodronate liposomes in mice, efficacy of anti-CD142 mAb therapy to prevent breast carcinoma outgrowth and metastases was reduced, indicating an important contribution of macrophages in mAb-dependent killing of tumor cells. TAMs may therefore represent promising candidates to target in anti-tumor mAb immunotherapy. Several studies supported the role of macrophages as effector cells in mAb therapy of cancer12,27-32. Human macrophages express the activating receptors FcγRI, FcγRIIa and FcγRIIIa as well as the inhibitory FcγRIIb5-7. Similarly, the activating receptors FcγRI, III and IV, and the inhibitory receptor FcγRII are expressed on mouse macrophages. It was previously demonstrated that efficacy of anti-tumor mAb therapy was increased in mice that were deficient in FcγRII13, which can not be attributed to NK cell-mediated ADCC, as these cells do not express FcγRII. Instead, monocytes and macrophages, which express activating Fc receptors as well as FcγRII, were likely involved as most prominent effector cell populations in mAb-mediated tumor elimination in vivo. We furthermore previously showed that liver metastases outgrowth after challenge with the gp75-expressing mouse melanoma cell line B16F10 was prevented by anti-gp75 (TA99) mAb treatment via a complement-independent manner12. However, mAb therapy was ineffective in FcR γ-chain knock out mice that lack all activating Fc receptors. By contrast, TA99 mAb treatment was successful in FcγRI-/-, FcγRIII-/-, double FcγRI-/-/III-/-, and triple FcγRI-/-/II-/-/III-/- mice, or after blocking of FcγRIV. Only when FcγRI and FcγRIV were simultaneously inhibited, TA99 mAb treatment did not inhibit development of liver metastases, suggesting an overlap in function for these receptors. As such, a prominent role for the mononuclear phagocyte network (monocytes and macrophages) in tumor elimination is indicated, as monocytes and macrophages are the only cells in mice expressing both FcγRI and FcγRIV5-7. Additionally, macrophage depletion decreased survival of SCID mice that had been engrafted with a Hodgkin-derived cell line, and which were treated with anti-CD30 mAbs. Removal of NK cells did not significantly influence therapeutic efficacy32. In a xenograft model of non-Hodgkin lymphoma, anti-CD40 mAb therapy was ineffective when macrophages were depleted31. Elimination of either NK cells or neutrophils

TABLE 1. Interactions of Fcγ receptors with different IgG isotypes (reviewed in6,7).

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did not affect therapeutic efficacy. Moreover, mutant anti-CD40 mAbs that lacked the ability to bind to Fc receptors were ineffective in protecting against tumor development. The importance of monocytes and/or macrophages in anti-CD20 mAb–mediated tumor elimination in mice was demonstrated, as well. Depletion of B cells by anti-murine CD20 mAbs was strictly dependent on the mononuclear phagocyte network, and required expression of activating Fcγ receptors33. Depletion of macrophages by clodronate furthermore abrogated the ability of rituximab to eradicate B cells in a human CD20 transgenic mouse model34. When mice were challenged with syngeneic B cell lymphoma cells, treatment with anti-murine CD20 mAbs did not prevent tumor development in mice lacking activating Fcγ receptors30. Moreover, when mice were injected with lymphoma cells after depletion of macrophages by clodronate liposomes, comparable poor survival rates were observed after either anti-CD20 mAb or control isotype mAb treatment. By contrast, mice receiving anti-CD20 mAb without clodronate liposomes (thus macrophages were present) survived a challenge with tumor cells. Mutant anti-CD20 mAbs that did not interact with C1q were still effective, indicating that survival of lymphoma-bearing mice after mAb therapy was not dependent on complement activation, but on tumor cell elimination by macrophages via Fcγ receptor-mediated processes. It was recently demonstrated that an IL-10–producing B cell subset (B10 cells) reduced efficacy of anti-CD20 mAb therapy of lymphoma-bearing mice through inhibition of monocyte/macrophage activation, which was overcome by treatment with a toll like receptor (TLR)3 agonist29. Since clinical responses to rituximab therapy correlate with polymorphisms in human FcγRIIa, which is expressed on myeloid effector cells, but not on NK cells, a role for macrophages in B cell lymphoma depletion after anti-CD20 mAb treatment of patients is supported14.

2.1. Mechanisms of tumor cell killing by the mononuclear phagocyte network. Thus, monocytes and macrophages likely play an important role in mAb-mediated tumor clearance in an Fcγ receptor dependent way, but the mechanism(s) of killing has not yet been completely elucidated. The mechanism most often described as effector mechanism induced by mAbs is ADCC18,35-38, which involves degranulation of the effector cell thereby lysing target cells. ADCC is primarily attributed to NK cells, but it has been suggested that monocytes and macrophages can also mediate ADCC37,39. Additionally, macrophages can phagocytose tumor cells in the presence of mAbs, which is referred to as antibody-dependent phagocytosis (ADCP). Of note, ADCC and ADCP have been used interchangeable to describe antibody-mediated killing of tumor cells by macrophages in some manuscripts in the past. Because ADCC is addressed by Matthias Peipp et al. in this issue of Methods, we will focus on ADCP in the next paragraphs.

2.1.1. Antibody-dependent phagocytosis.The name macrophage is derived from the Greek words makros (large) and phagein (eat). As such, one of their main functions is to phagocytose and digest cellular debris and pathogens. Although macrophages can engulf these particles in a nonspecific fashion via pattern recognition receptors (PRRs), such as TLR and C-type lectins, uptake is much more efficient after opsonization with antibodies (and/or complement factors), as macrophages express complement and Fc receptors. Most mAbs that have been used to study enhanced phagocytosis of tumor cells were of the IgG subclass. Enhanced in vitro ADCP with human effector cells has been described for

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1the tumor-associated antigens carcinoembryonic antigen (CEA), HER-2, epithelial cell adhesion molecule (Ep-CAM), human epithelial mucin (MUC)-1, CD20, CD30 and CD4031,32,36,40-45. Because IgG can also bind to the inhibitory FcgRIIb receptor on macrophages, it was furthermore investigated whether targeting of specific Fc receptors with bispecific antibodies (BsAb) improved ADCP. A BsAb recognizing the high affinity FcγRI on human macrophages and CD30 on lymphoma cells was able to effectively induce ADCP46. A BsAb targeting the IgA Fc receptor FcαRI instead of FcγRI was equally effective. BsAb targeting FcγRI and HER-2 or BsAb targeting FcαRI and HER-2 were furthermore able to induce phagocytosis of human HER-2-overexpressing breast carcinoma cells by macrophages45,47. Similarly, IgA anti-Ep-CAM was able to induce ADCP, but proved far less effective compared to an IgG anti-Ep-CAM counterpart42. We previously demonstrated that ADCP by liver macrophages (Kupffer cells) contributed to the in vivo elimination of circulating tumor cells in a rat model48. However, when rats were treated with high doses mAbs, monocytes were recruited as effector cells as well, in which case ADCC likely contributed to therapeutic efficacy. When macrophages were depleted with clodronate liposomes, mAb therapy was unsuccessful in preventing liver metastases development, strongly supporting the involvement of the mononuclear phagocytic network of the liver. Phagocytosis of T lymphoma cells by Kupffer cells after treatment with an anti-fibronectin receptor mAb was reported as well, which was in part responsible for prolonged survival of mice49.

2.1.2. Enhancement of ADCP by macrophages.Several different strategies have been employed to increase the ability of macrophages to mediate ADCP. It was demonstrated that cytokines regulate Fcγ receptor expression, which correlated with mAb-mediated uptake50. For instance, human monocyte-derived macrophages that were grown in the presence of interferon-γ were more efficient in inducing ADCP in the presence of FcγRI BsAb, whereas granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated macrophages had increased uptake of tumor cells in the presence of FcαRI BsAb51. Recently, it was shown that human macrophages, which were differentiated in the presence of macrophage-colony stimulating factor (M-CSF), phagocytosed more lymphoma cells in the presence of rituximab, compared to macrophages that had been induced with GM-CSF52. Alternatively, engineered mAbs with mutations in their Fc tails have been described, which influence binding to Fcγ receptors. For instance, anti-Ep-CAM mAbs were described with engineered Fc that increased the affinity for FcγRIIa up to seventy-fold, which augmented ADCP of LS180 adenocarcinoma cells by human macrophages53. Similarly, engineered anti-CD19, anti-CD40 or anti-HM1.24 mAbs have been documented that increased phagocytosis of B-lymphoma, leukemia, and multiple myeloma cell lines54-56. A mutated variant of trastuzumab, in which the Fc was aglycosylated resulted in a seventy-five percent enhancement of ADCP of tumor cells with low- to medium expression levels of HER-257. It was demonstrated that tumor cells, which express CD47 are more resistant to antibody-induced killing in mice58. CD47 interacts with the inhibitory receptor signal regulatory protein-α (SIRPα; CD172a) that is expressed on myeloid cells, such as macrophages, dendritic cells (DCs) and granulocytes59. Blocking CD47 with mAbs enabled phagocytosis of tumor cells by both human and mouse macrophages60,61. Furthermore, addition of blocking anti-CD47 mAbs in combination with rituximab increased ADCP of non-Hodgkin lymphoma cells by macrophages when compared to addition of rituximab alone62. Co-treatment with anti-CD47 antibody and rituximab of

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mice that had been engrafted with human non-Hodgkin lymphoma cells eliminated lymphoma cells and led to cure, which was dependent on macrophages63. Similarly, TA99 mAb therapy was more effective in preventing melanoma lung metastases in mice with a mutant SIRPα, which lack the intracellular tail (and thus the inhibitory signal)58. Thus, anti-tumor mAb therapy is more effective when additionally, the CD47-SIRPα pathway is blocked.

3. Methods to detect phagocytosis. Techniques that are frequently used to analyze (mAb-mediated) tumor cell killing in vitro are radioactive chromium or fluorescence release assays13,41,58,61,64,65. To this end, target cells are labeled with either radioactive chromium (51Cr) or a fluorescent probe. Subsequently, labeled target cells are incubated for a period of time with effector cells (e.g. NK cells, cytotoxic T cells, monocytes or neutrophils) with or without mAbs, after which supernatants are collected and analyzed for the presence of 51Cr or fluorescence, which is a measure of the amount of cell death. These assays are however not suitable to study ADCP, as phagocytosis of complete tumor cells by macrophages will not result in release of 51Cr or fluorescence probes into the supernatant. Several other techniques can be employed to analyze this process, including flow cytometry and different types of microscopy5. In the next paragraphs we describe the advantages and disadvantages of current techniques to study ADCP.

3.1 Experimental setupOverall protocols to perform phagocytosis experiments are similar, with slight variations26,29,31,36,41,61,64,65. First, macrophages need to be obtained. Although primary macrophages can be isolated from peritoneal cavities or though lung lavages29, most protocols include the culture of macrophages from either peripheral blood monocytes (human) or bone marrow (mouse and rats). Culture schemes usually include the addition of recombinant M-CSF or conditioned medium from M-CSF secreting cells for 6-8 days. Alternatively, GM-CSF or IFN-γ has been added to macrophage cultures36,41,52,64.Second, both macrophages and tumor cells are labeled with fluorescent probes. This can vary from transient or stable expression with green or red fluorescent proteins (GFP or RFP), staining with membrane dyes, such as DiI (red), DiO (green), DiB (blue) or PKH26 (red) and PKH67 (green), or intracellular staining with CFSE or CFDA SE (green). Alternatively, cell specific markers have been used for detection (e.g. conjugated anti-CD14 mAbs to detect human macrophages, F4/80 mAbs to stain mouse macrophages, or mAbs specific for tumor-associated antigens to detect tumor cells). Third, macrophages and target cells are co-cultured for different time periods, which can range from 30 minutes to several days. Additionally, different effector to target (E:T) ratios have been described that typically vary from 1 effector cell to 1 target cell (E:T=1:1) up to an E:T of 15:1. Different concentrations of anti-tumor mAbs are added during the incubation period, although pre-opsonization of tumor cells for 30 minutes-1 hour has been described as well.

3.2 Flow cytometryOne of the methods that is mostly used to determine ADCP is flow cytometry. Fluorescence based cell sorting (FACS) allows the measurement of individual cells in a flow system that delivers single cells past a point of detection, after which scattered

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light and fluorescence of different wavelengths are quantified. The great strength of this technique is the ability to record characteristics of large numbers of single cells in a short time period. The possibilities of this technique have recently been extensively addressed in a special edition of Methods66. After labeling of macrophages with one fluorescent dye, and tumor cells with another, three different populations can be distinguished with flow cytometry, which represent 1) macrophages, 2) tumor cells and 3) double positive cells that are assumed to be macrophages that have bound or taken up (parts of) tumor cells (Figure 2). One advantage of flow cytometry is that this method has a fast quantitative readout. Directly after harvesting, cells can be analyzed, allowing rapid screening of different culture conditions, such as incubation times, different mAb concentrations, or addition of supplementary factors that can promote or inhibit ADCP. However, it is not possible to make a distinction between tumor cells that are bound to macrophages, versus tumor cells that have been phagocytosed, as both will be detected as double-positive cells. Additionally, it is difficult to distinguish between macrophages that have phagocytosed one or multiple tumor cells or only part of a tumor cell. (Confocal) microscopy can be used to investigate the latter, but quantification with this technique is labor intensive. A relatively recent development is the use of imaging flow cytometers, which combine capabilities of microscopy and flow cytometry in a single platform. The ImageStream® system can acquire multiple images of single cells in flow that comprise of a side-scatter (darkfield) and transmitted light (bright field) images and multiple fluorescence images (https://www.amnis.com/imagestream.html). This allows generation of scatter plots and histograms comparable to standard flow cytometry, but images also provide additional information like cell size and shape, heterogeneity of probe distribution, intracellular or membrane-bound location of fluorescent dyes, and co-localization of different fluorescent probes. One of the

FIGURE 2. ADCP measured with standard flow cytometry.(A) Gating of different cell populations. DiI-labeled macrophages are depicted on the X-axis, whereas DiO-labeled Daudi lymphoma cells are shown on the Y-axis. Macrophages and tumor cells have been incubated with anti-CD20 mAb ofatumumab (left panels) or isotype control mAbs (right panels) with an E:T ratio of 15:1 (upper panels) or 5:1 (lower panels). (B) Quantification of remaining Daudi cells, macrophages and double positive macrophages that have bound or phagocytosed tumor cells as well as macrophages that were involved in trogocytosis. Data represents triplicates.

A B

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major advantages of this technique is that quantification of ADCP, including means, medians, standard deviation and statistics can be performed. In addition, cell images can be visually analyzed by generating masks with defined visual criteria, resulting in identification and quantification of tumor cell uptake, starting with cell-to-cell contact, and progressing into complete phagocytosis and degradation (Figure 3). Furthermore, it is possible to quantify the number of tumor cells that have been taken up. Analyses of data obtained by imaging flow cytometry is however quite laborious when compared to standard flow cytometry. Furthermore, as with standard flow cytometry, data will always represent snapshots of the process of phagocytosis and degradation, even when time courses are executed. 3.3 Imaging

FIGURE 3. ADCP measured with imaging flow cytometry (ImageStream).The experiment was performed as described in the supplementary Materials and Methods. Macrophages were incubated with Daudi lymphoma cells in the presence of ofatumumab for 1 hour, after which samples were analyzed on the ImageStream (AMNIS) and supplied software (IDEAS). (A) Highly double positive cells (DiO-labeled tumor cells on Y axis and F4/80+ macrophages on X-axis) were gated (upper graph). This population was subsequently analyzed in depth by generating masks on the cells, as shown in the lower bi-variate plot. On the X-axis the area of the tumor cell minus the area of the macrophage is depicted, which indicates that the score is negative (-) when a tumor cell is smaller than a macrophage. On the Y-axis phagocytosis is shown. When the fluorescence signal of a tumor cell is inside the mask of the fluorescence signal of a macrophage a positive (+) score is obtained. When no tumor cell uptake is observed, cells are assigned a negative score. Different gates are shown to illustrate the different populations that can be distinguished in the highly double positive cells. Gate 1 has a high phagocytosis score and a negative score for tumor to macrophage area comparison. Examples of images are shown in the 2 upper panels in (B). Panel 1 depicts a tumor cell that has been completely taken up by a macrophage, whereas panel 2 shows the beginning of degradation of a tumor cell within a macrophage or possibly trogocytosis. Panel 3 illustrates an example from gate 2. There is strong co-localization between a DiO-labeled tumor cell and a macrophage, and a highly negative score for area comparison. Furthermore, the image demonstrates that in addition to uptake of a DiO-labeled tumor cell, also a DiB- labeled tumor cell has been phagocytosed. An example from gate 3 is shown in panel 4, which demonstrates complete engulfment of a tumor cell by a macrophage of similar size. Finally the lower 2 panels are examples of events in gate 4. Panel 5 depicts complete lack of internalization, which indicates that the tumor cell is bound, and not phagocytosed. Moreover, the bright field image shows that the macrophage is presumably in apoptosis as indicated by membrane blebbing. In panel 6 partial phagocytosis is detected, which is reflected by one tumor cell that has been taken up, and one tumor cell that is in the process of being phagocytosed.

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1Microscopy is a good technique to investigate tissue morphology and processes in the context of the environment, which is lost during preparation of single cells for flow cytometry67. Traditional microscopy mostly provides snapshots from a process in time, but development of live cell imaging allows the visualization of phagocytosis over time without disrupting the morphology of the cells or tissues67. With this technique the whole process of phagocytosis can be observed, which can sometimes yield unexpected results. For instance, as can be observed in Figure 4 and supplementary video 1 and 2 the macrophage depicted with (1) is interacting with a tumor cell (indicated with an asterisk). After 20 minutes, a second macrophage starts interacting with the same tumor cell, which led to phagocytosis of the complete tumor cell after 30 minutes. Nonetheless, macrophage 1 has accumulated the membrane dye of the tumor cell, which suggests sampling of the tumor cells during probing. This may represent a process referred to as trogocytosis68-70. Trogocytosis can occur when effector cells bind to antigens on target cells, after which ligands and the surrounding membrane are transferred from target to effector cells. In vitro experiments with human cells suggested that trogocytosis may occur in cancer patients treated with rituximab, cetuximab, or trastumumab, as uptake of the target-antibody complex without phagocytosis of the target cell was observed68,69. Loss of rituximab-CD20 complexes without elimination of malignant B cells was also observed in patients with chronic lymphocytic leukemia that were treated with rituximab, suggesting that trogocytosis or shaving can occur in patients71. When translating live cell imaging results with data analysis with standard flow cytometry, both macrophage 1 and 2 would end up in the double positive population, even though only macrophage 2 has in fact taken up a tumor cell, whereas macrophage 1 was engaged in trogocytosis. Analysis with the ImageStream would suggest that macrophage 1 has phagocytosed a tumor cell, which is already degraded. Thus, a great advantage of live cell imaging is the accurate illustration of processes in time. Analysis is however, quite laborious, and not the most suitable for quantification of phagocytosis.

FIGURE 4. ADCP visualized with live cell microscopy. DiI-labeled macrophages (red) and DiB-labeled Daudi lymphoma cells (blue) were incubated with ofatumumab and followed in time. At the beginning of the recording macrophage 1 interacts (white arrow heads) with a tumor cell (indicated by asterisk). After 20 minutes of recording macrophage 2 starts to interact (yellow arrow heads) with the tumor cell, which results in phagocytosis at 30 minutes. Nonetheless, macrophage 1 has obtained blue dye, suggesting sampling of the tumor cells without actual phagocytosis (trogocytosis). The upper panels show overlays of bright field and fluorescence, whereas the other panels only show fluorescence (magnification of indicated area is shown in lower panels). For movies, scan QR-code for video 1 and 2.

Video 2Video 1

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4. Recruitment of neutrophils in mAb therapy of cancer4.1 mAb-mediated killing of tumor cells by neutrophilsNeutrophils are the most abundant circulating white blood cell population, and their numbers are easily amplified in vivo without the need for ex vivo expansion, by simply treating patients with granulocyte-colony stimulating factor (G-CSF) or GM-CSF72,73. Thus, an advantage of recruiting neutrophils for tumor therapy is the relative ease of obtaining a formidable source of cytotoxic effector cells (Figure 5). Neutrophils produce a plethora of cytotoxic molecules including proteases, oxidative metabolites, and defensins74. They have potent bactericidal and fungicidal activity, and it has been demonstrated that they can exert direct tumoricidal activity, as neutrophils were important for inducing tumor elimination in animal models75-78. Nonetheless, their overall ability to recognize and kill tumor cells is limited, which is however increased in the presence of anti-tumor mAbs79. The role of neutrophils as effector cells in current mAb therapies has not yet been established, but is was shown that neutrophil depletion decreased therapeutic activity of the clinically used anti-CD52 mAb alemtuzumab (Campath-1H) in a CD52+ xenograft tumor model80. Additionally, rituximab was less effective in reducing lymphoma development after depletion of neutrophils in a B-cell lymphoma model81. As such, the involvement of neutrophils as cytotoxic effector cells was suggested. Combination therapies including G-CSF and rituximab with or without additional chemotherapy (CHOP) were well tolerated in patients with B cell malignancies in Phase I or II studies, but no clinical benefit of G-CSF addition was yet observed82,83. Higher response rates were observed when patients with Follicular Lymphoma received GM-CSF and rituximab, which may have been a result of enhanced neutrophil numbers, although an effect on monocytes, macrophages and/or DCs cannot be excluded84.Neutrophils constitutively express FcγRIIa, FcγRIIIb, and FcαRI. Furthermore, FcγRI is upregulated after exposure to IFN-γ or G-CSF85,86. FcγRIIIb, which is a glycosyl-phosphatidylinositol-anchored receptor, is the most abundant Fcγ receptor on neutrophils, but probably not involved in efficient tumor cell cytotoxicity87. Antibody-dependent killing of tumor cells in the absence of G-CSF is likely mediated through FcγRIIa that bears an immunoreceptor tyrosine-based activation motif in its cytoplasmic tail88. The FcγRIIa polymorphism plays a role in cytotoxic ability in the presence of an IgG2 anti-EGFR mAb (panitumumab), but not when an IgG1 anti-EGFR mAb (zalutumumab) was added89. Furthermore, blocking CD47-SIRPα interactions with F(ab’)2 anti-CD47 resulted in enhanced killing of breast carcinoma cells by neutrophils in the presence of the anti-HER-2 mAb Herceptin58. When patients are treated with G-CSF, neutrophils express FcγRI. Therefore, specific targeting of FcγRI has been proposed, as this may overcome potential inhibitory signals through FcγRIIb when IgG mAb are employed13,90. FcγRI BsAb were efficient in recruiting neutrophils as effector cells88. Furthermore, FcγRI-expressing neutrophils of G-CSF treated patients were more effective in mediating mAb-mediated tumor cell killing than neutrophils from healthy donors that lack FcγRI90. A multitude of FcγRI BsAb have now been described that are directed against different tumor antigens (including HLA class II, and the HLA class II variants Lym-1 and Lym-2 for targeting lymphoma; EGFR, Ep-CAM or HER-2 for treating epithelial malignancies; and bombesin which binds to small cell carcinoma of the lung)91. Efficacy of FcγRI BsAb in vivo was shown in FcγRI transgenic mice, but overall outcome in phase I/II clinical trials was disappointing, which may have been due to the short half life of BsAb92-96.

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1FcαRI has previously been identified as alternative target molecule for antibody-based tumor therapy. It has now been shown that FcαRI in fact represents the most potent Fc receptor to induce antibody-mediated tumor cell killing by neutrophils, which has been described for a plethora of tumor antigens, including Ep-CAM (colon carcinoma), HER-2 (breast carcinoma), EGFR (epithelial and renal cell carcinoma), HLA class II and CD20 (B cell lymphoma), CD30 (B and T cell lymphoma), and CEA42,97-100. Additionally, it was demonstrated that immature bone marrow neutrophils, which are mobilized during G-CSF therapy, effectively mediated antibody-mediated cytotoxicity via FcαRI, but not through FcγRI101,102. A combination therapy that includes G-CSF and targeting of FcαRI on neutrophils may therefore improve clinical responses compared to earlier disappointing trials in which FcγRI BsAb were used. Furthermore, FcαRI is the only Fc receptor that mediated neutrophil recruitment, as leukotriene B4 (LTB4) - a potent neutrophil chemoattractant – was released after FcαRI cross-linking103. Targeting of FcαRI, but not of Fcγ receptors resulted in neutrophil accumulation in 3-dimensional tumor colonies, which has been demonstrated for HER-2 (breast carcinoma), EGFR (epithelial carcinoma) and Ep-Cam (colon carcinoma)97,102,104. Moreover, targeting FcαRI led to cross-talk between neutrophils and endothelial cells, as the latter released CXCL8, which is a prototypic neutrophil chemokine104. Subsequently, increased neutrophil migration into and destruction of tumor colonies was observed.Thus, FcαRI represents a promising candidate target molecule. In vivo studies in which FcαRI was targeted have however been hampered due to unavailability of suitable models, as mice lack FcαRI105. Moreover, production of sufficient amounts of human IgA anti-tumor associated antigens was difficult in the past. With the establishment of human FcαRI transgenic mice106,107, and improvement of human IgA antibody production and purification techniques108,109, progress of therapeutic IgA mAb development should be greatly facilitated in the near future. The first paper, demonstrating the in vivo efficacy of IgA anti-tumor mAbs was recently published, in which it was reported that injection of naked plasmid DNA encoding anti-CD20 IgA2 mAbs effectively prevented development of B cell lymphoma110.

4.2 Mechanisms of neutrophil-induced mAb-dependent tumor killing. The mechanisms underlying neutrophil mediated antibody-dependent tumor cell are not yet fully established. Neutrophils possess a diverse arsenal of toxic components, required for their prominent antimicrobial functions (Figure 5A)111. Neutrophils additionally induce a respiratory burst, resulting in generation of reactive oxygen species (ROS). It was shown that release of defensins - that are present in neutrophil azurophilic granules -, rapidly damaged tumor cells, which was enhanced in the presence of H2O2

112. However, neutrophils from patients with chronic granulomatous disease, which cannot produce ROS, effectively induced antibody-mediated tumor cell killing113,114. ROS scavengers or inhibitors did not inhibit neutrophil-mediated tumor cell killing in the presence of mAbs either, supporting that ROS is not crucial for tumor cell killing. It was recently described that neutrophils can release neutrophil extracellular traps (NETs) after stimulation, which constitutes another antimicrobial mechanism of neutrophils115. NETs are composed of nuclear components like DNA and histones and contain distinct granular and cytoplasmic proteins, such as elastase and MPO115. They form a mesh that traps micro-organisms, but it has been described that NETs have toxic effects on host cells as well. For instance, exposure to MPO and histones in NETs induced epithelial and endothelial cell death116. We observed that anti-tumor

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IgA mAbs resulted in NETs release, although it remains to be established whether this will result in killing of tumor cells (own unpublished data).Close contact between tumor cells and neutrophils - referred to as ‘immunological synapses’ or ‘cytotoxic synapses’ - is essential for the induction of tumor cell killing117. Complement receptor 3 (CR3) is essential for intimate spreading of neutrophils on tumor cells. Cytotoxic synapses could not be formed by CR3-deficient neutrophils, with concomitant absence of tumor cell killing107,117. In vivo presence of cytotoxic synapses between neutrophils and tumor cells in the presence of mAbs was recently shown37. It was furthermore demonstrated that CR3 cooperates with FcγRIIIb in initiating respiratory burst in human neutrophils in vitro118. The capacity of neutrophils to initiate apoptosis in tumor cells is debatable. NK cells and cytotoxic T lymphocytes (CTL) use amongst others granzymes and perforin to induce apoptosis in targets cells. The presence of these molecules in neutrophils, as reported by some groups, is controversial, because other groups were unable to confirm these findings119-122. Nonetheless, apoptosis of HER-2-positive tumor cells was shown after a 20 hour incubation period in the presence of neutrophils and anti-HER-2 mAbs113,123. We recently identified two non-apoptotic pathways that are induced in tumor cells by neutrophils. Induction of necrosis was observed in a small population of tumor cells, whereas the majority displayed autophagic characteristics124. Autophagy is a process of self-digestion, which will result in generation of energy. As such, it is mostly regarded as a cell survival mechanism, but may become an alternative cellular suicide pathway under excessive stress conditions125. Whether autophagic cell death

FIGURE 5. Schematic representation of neutrophils as effector cells in tumor therapy. (A) Neutrophils can release multiple toxic components and NETs, which may lead to tumor cell killing. Cytotoxic abilities are increased in the presence of anti-tumor mAbs (especially IgA). Furthermore, the number of neutrophils expands rapidly after treatment with G-CSF or GM-CSF. (B) Neutrophils play a role in innate and adaptive immune responses. Upper part: Through secretion of chemokines, cytokines and growth factors several other immune cells can be activated and recruited. Lower part: cross-talk between neutrophils and other immune cells has been reported (see also main text for more details).

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1is a true separate pathway or whether characteristics of autophagy are involved in a yet uncharacterized novel cell death pathway is still debated126. Nevertheless, targeting neutrophils may represent a desirable strategy to trigger non-apoptotic cell death in tumor cells that are resistant for apoptosis induction.

4.3 Neutrophils and regulation of adaptive immune responses. An additional advantage of recruiting neutrophils for mAb therapy of cancer is their ability to regulate and activate adaptive immune responses (Figure 5B)127. Neutrophils are generally considered terminally differentiated cells with limited ability for biosynthesis. However, it is becoming clear that they can produce a multitude of cytokines, chemokines, and angiogenic factors, which influence other immune and non-immune cells127. Neutrophils may produce fewer molecules per cell compared to other cells, but their sheer number can contribute significantly to the total amount of inflammatory mediators and angiogenic factors. In fact, cross-talk between neutrophils and several other immune cells was recently reported. For instance, release of CCL2 and CCL20 by neutrophils led to T helper 17 (Th17) cell recruitment, which in turn secreted CXCL8128. Additionally, IL-17 release induces production of G-CSF by epithelial cells. Depletion of neutrophils in mouse tumor models led to decreased recruitment of CD4+ T cells and reduced CD8+ T cell activation, supporting the role of neutrophils in T cell function78,129. With respect to mAb cancer therapy it is also noteworthy that neutrophils promote NK cell proliferation, cytotoxic ability and survival130,131. NK cells subsequently release IFN-γ and GM-CSF, supporting neutrophil activation and survival. Interactions between neutrophils and DCs have been demonstrated as well, and as such it was proposed that neutrophils act as danger sensors by communicating presence of inflammation to DCs132,133. This will result in DC maturation via TNF-α release by neutrophils, which will subsequently lead to IL-12 production by DCs, enhanced DC-induced T cell proliferation, and polarization into a Th1 phenotype133,134. It was furthermore shown that neutrophils transfer antigens to DCs, leading to specific T cell responses135.

5. In conclusion Myeloid cells represent potent effector cells for mAb therapy of cancer. The involvement of these cells in mAb-based therapeutic strategies is supported by several animal studies. Furthermore, a role for myeloid cells in rituximab treatment of patients is supported, since polymorphisms in human FcγRIIa – which is expressed on monocytes, macrophages and neutrophils, but not on NK cells – correspond with clinical responses. This is likely mediated by the mononuclear phagocytic network, as these cells can efficiently kill or phagocytose tumor cells in the presence of IgG mAbs. In vitro phagocytosis can be studied with flow cytometry or microscopy. Standard flow cytometry has been used frequently, and is an easy method to study optimal mAb concentrations with or without additional stimuli, E:T ratios and time courses. However, it will not provide details about binding versus uptake, the number of cells that have been phagocytosed, whether trogocytosis has occurred or the state of degradation. The use of imaging flow cytometry can provide more insights in these processes, and as such is more suitable to study phagocytosis. Nonetheless, all flow cytometry- based methods will only provide snapshots of a process in time. Furthermore, as single cells are required, relation of cells with the environment is lost. To investigate the latter, microscopy is more suitable. Furthermore, live cell microscopy will allow in-depth analyses of processes over time. It is however less suitable for quantification of phagocytosis.

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Neutrophils are more effective in the presence of IgA mAbs. In fact, the contribution of neutrophils to current clinical responses is not clear, as IgA mAbs have not yet been used in clinical trials. Nonetheless, neutrophils represent attractive effector cells, as they possess potent cytotoxicity, are abundantly present and easily mobilized, and have the ability to induce adaptive immune responses. Furthermore, killing of tumor cells via targeting FcαRI on neutrophils is not dependent on complement, which is beneficial when tumors express membrane bound complement regulators protecting them from CDC136. An additional advantage is the induction of non-apoptotic cell death after targeting neutrophil FcαRI, which may represent an attractive strategy for killing tumor cells, which have mutations in apoptotic pathways124. Thus, recruiting myeloid cells in mAb therapy may augment clinical efficacy, especially when their cytotoxic abilities are further enhanced by modulation of mAbs, co-treatment with cytokines, or blocking CD47-SIRPα interactions, which needs to be established in further studies.

AcknowledgementsThe authors like to thank R. Korthouwer, MSc for expert help with experimental procedures and Genmab (Utrecht, The Netherlands) for kindly providing ofatumumab and isotype mAbs.

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1Reference list1. Scott, A.M., Wolchok, J.D. & Old, L.J. Antibody therapy of cancer. Nature reviews 12, 278-287 (2012).2. Weiner, L.M., Surana, R. & Wang, S. Monoclonal antibodies: versatile platforms for cancer immunotherapy.

Nat Rev Immunol 10, 317-327 (2010).3. Benvenuti, S., et al. Oncogenic activation of the RAS/RAF signaling pathway impairs the response

of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies. Cancer research 67, 2643-2648 (2007).

4. Racila, E., et al. A polymorphism in the complement component C1qA correlates with prolonged response following rituximab therapy of follicular lymphoma. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 6697-6703 (2008).

5. Golay, J. & Introna, M. Mechanism of action of therapeutic monoclonal antibodies: promises and pitfalls of in vitro and in vivo assays. Archives of biochemistry and biophysics 526, 146-153 (2012).

6. Bruhns, P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood 119, 5640-5649 (2012).

7. Hogarth, P.M. & Pietersz, G.A. Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nat Rev Drug Discov 11, 311-331 (2012).

8. Nimmerjahn, F. & Ravetch, J.V. FcgammaRs in health and disease. Current topics in microbiology and immunology 350, 105-125 (2011).

9. Clynes, R., Takechi, Y., Moroi, Y., Houghton, A. & Ravetch, J.V. Fc receptors are required in passive and active immunity to melanoma. Proceedings of the National Academy of Sciences of the United States of America 95, 652-656 (1998).

10. Bevaart, L., et al. The high-affinity IgG receptor, FcgammaRI, plays a central role in antibody therapy of experimental melanoma. Cancer research 66, 1261-1264 (2006).

11. Nimmerjahn, F. & Ravetch, J.V. Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science (New York, N.Y 310, 1510-1512 (2005).

12. Otten, M.A., et al. Experimental antibody therapy of liver metastases reveals functional redundancy between Fc gammaRI and Fc gammaRIV. J Immunol 181, 6829-6836 (2008).

13. Clynes, R.A., Towers, T.L., Presta, L.G. & Ravetch, J.V. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature medicine 6, 443-446 (2000).

14. Weng, W.K. & Levy, R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol 21, 3940-3947 (2003).

15. Musolino, A., et al. Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer. J Clin Oncol 26, 1789-1796 (2008).

16. Bibeau, F., et al. Impact of Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms and KRAS mutations on the clinical outcome of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan. J Clin Oncol 27, 1122-1129 (2009).

17. Zamai, L., et al. NK cells and cancer. J Immunol 178, 4011-4016 (2007).18. Vivier, E., et al. Innate or adaptive immunity? The example of natural killer cells. Science (New York, N.Y

331, 44-49 (2011).19. Hatjiharissi, E., et al. Increased natural killer cell expression of CD16, augmented binding and ADCC

activity to rituximab among individuals expressing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism. Blood 110, 2561-2564 (2007).

20. Mantovani, A., Biswas, S.K., Galdiero, M.R., Sica, A. & Locati, M. Macrophage plasticity and polarization in tissue repair and remodelling. The Journal of pathology 229, 176-185 (2013).

21. Mosser, D.M. & Edwards, J.P. Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8, 958-969 (2008).

22. Leek, R.D., et al. Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer research 56, 4625-4629 (1996).

23. Forssell, J., et al. High macrophage infiltration along the tumor front correlates with improved survival in colon cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 1472-1479 (2007).

24. Zhou, Q., et al. The density of macrophages in the invasive front is inversely correlated to liver metastasis in colon cancer. Journal of translational medicine 8, 13 (2010).

25. Lewis, C.E. & Pollard, J.W. Distinct role of macrophages in different tumor microenvironments. Cancer research 66, 605-612 (2006).

26. Grugan, K.D., et al. Tumor-associated macrophages promote invasion while retaining Fc-dependent anti-tumor function. J Immunol 189, 5457-5466 (2012).

27. De Palma, M. & Lewis, C.E. Macrophage regulation of tumor responses to anticancer therapies. Cancer

34

Chapter 1

cell 23, 277-286 (2013).28. Fridlender, Z.G., et al. Using macrophage activation to augment immunotherapy of established tumours.

British journal of cancer 108, 1288-1297 (2013).29. Horikawa, M., Minard-Colin, V., Matsushita, T. & Tedder, T.F. Regulatory B cell production of IL-10 inhibits

lymphoma depletion during CD20 immunotherapy in mice. The Journal of clinical investigation 121, 4268-4280 (2011).

30. Minard-Colin, V., et al. Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage FcgammaRI, FcgammaRIII, and FcgammaRIV. Blood 112, 1205-1213 (2008).

31. Oflazoglu, E., et al. Macrophages and Fc-receptor interactions contribute to the antitumour activities of the anti-CD40 antibody SGN-40. British journal of cancer 100, 113-117 (2009).

32. Oflazoglu, E., et al. Macrophages contribute to the antitumor activity of the anti-CD30 antibody SGN-30. Blood 110, 4370-4372 (2007).

33. Uchida, J., et al. The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy. The Journal of experimental medicine 199, 1659-1669 (2004).

34. Gong, Q., et al. Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy. J Immunol 174, 817-826 (2005).

35. Houot, R., Kohrt, H.E., Marabelle, A. & Levy, R. Targeting immune effector cells to promote antibody-induced cytotoxicity in cancer immunotherapy. Trends in immunology 32, 510-516 (2011).

36. Bologna, L., et al. Ofatumumab is more efficient than rituximab in lysing B chronic lymphocytic leukemia cells in whole blood and in combination with chemotherapy. J Immunol 190, 231-239 (2013).

37. Hubert, P., et al. Antibody-dependent cell cytotoxicity synapses form in mice during tumor-specific antibody immunotherapy. Cancer research 71, 5134-5143 (2011).

38. Teeling, J.L., et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 104, 1793-1800 (2004).

39. te Velde, A.A., de Waal Malefijt, R., Huijbens, R.J., de Vries, J.E. & Figdor, C.G. IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10. J Immunol 149, 4048-4052 (1992).

40. Akewanlop, C., et al. Phagocytosis of breast cancer cells mediated by anti-MUC-1 monoclonal antibody, DF3, and its bispecific antibody. Cancer research 61, 4061-4065 (2001).

41. Ashraf, S.Q., et al. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis. British journal of cancer 101, 1758-1768 (2009).

42. Huls, G., et al. Antitumor immune effector mechanisms recruited by phage display-derived fully human IgG1 and IgA1 monoclonal antibodies. Cancer research 59, 5778-5784 (1999).

43. Lefebvre, M.L., Krause, S.W., Salcedo, M. & Nardin, A. Ex vivo-activated human macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism of antibody-dependent cellular cytotoxicity and impact of human serum. J Immunother 29, 388-397 (2006).

44. Rafiq, S., et al. Comparative assessment of clinically utilized CD20-directed antibodies in chronic lymphocytic leukemia cells reveals divergent NK cell, monocyte, and macrophage properties. J Immunol 190, 2702-2711 (2013).

45. Watanabe, M., et al. Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210. Breast cancer research and treatment 53, 199-207 (1999).

46. Sundarapandiyan, K., et al. Bispecific antibody-mediated destruction of Hodgkin’s lymphoma cells. Journal of immunological methods 248, 113-123 (2001).

47. van Egmond, M., Hanneke van Vuuren, A.J. & van de Winkel, J.G. The human Fc receptor for IgA (Fc alpha RI, CD89) on transgenic peritoneal macrophages triggers phagocytosis and tumor cell lysis. Immunology letters 68, 83-87 (1999).

48. van der Bij, G.J., et al. Experimentally induced liver metastases from colorectal cancer can be prevented by mononuclear phagocyte-mediated monoclonal antibody therapy. Journal of hepatology 53, 677-685 (2010).

49. Ito, M., et al. In vivo tumouricidal effects of LAD-1 monoclonal antibody on murine RL-male-1 lymphoma mediated by enhanced phagocytosis. Clinical and experimental immunology 141, 54-61 (2005).

50. Guyre, P.M., Morganelli, P.M. & Miller, R. Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes. The Journal of clinical investigation 72, 393-397 (1983).

51. Keler, T., et al. Differential effect of cytokine treatment on Fc alpha receptor I- and Fc gamma receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages. J Immunol 164, 5746-5752 (2000).

35

Chapter 1

152. Leidi, M., et al. M2 macrophages phagocytose rituximab-opsonized leukemic targets more efficiently

than m1 cells in vitro. J Immunol 182, 4415-4422 (2009).53. Richards, J.O., et al. Optimization of antibody binding to FcgammaRIIa enhances macrophage

phagocytosis of tumor cells. Molecular cancer therapeutics 7, 2517-2527 (2008).54. Horton, H.M., et al. Potent in vitro and in vivo activity of an Fc-engineered anti-CD19 monoclonal

antibody against lymphoma and leukemia. Cancer research 68, 8049-8057 (2008).55. Horton, H.M., et al. Fc-engineered anti-CD40 antibody enhances multiple effector functions and exhibits

potent in vitro and in vivo antitumor activity against hematologic malignancies. Blood 116, 3004-3012 (2010).

56. Tai, Y.T., et al. Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function. Blood 119, 2074-2082 (2012).

57. Jung, S.T., et al. Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcgammaRIIa affinity and selectivity. ACS chemical biology 8, 368-375 (2013).

58. Zhao, X.W., et al. CD47-signal regulatory protein-alpha (SIRPalpha) interactions form a barrier for antibody-mediated tumor cell destruction. Proceedings of the National Academy of Sciences of the United States of America 108, 18342-18347 (2011).

59. Chao, M.P., Weissman, I.L. & Majeti, R. The CD47-SIRPalpha pathway in cancer immune evasion and potential therapeutic implications. Current opinion in immunology 24, 225-232 (2012).

60. Kim, D., et al. Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells. Leukemia 26, 2538-2545 (2012).

61. Willingham, S.B., et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proceedings of the National Academy of Sciences of the United States of America 109, 6662-6667 (2012).

62. Chao, M.P., et al. Anti-CD47 antibody synergizes with rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. Cell 142, 699-713 (2010).

63. Chao, M.P., et al. Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy. Blood 118, 4890-4901 (2011).

64. Brezski, R.J., Oberholtzer, A., Strake, B. & Jordan, R.E. The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge. mAbs 3, 558-567 (2011).

65. McEarchern, J.A., et al. Engineered anti-CD70 antibody with multiple effector functions exhibits in vitro and in vivo antitumor activities. Blood 109, 1185-1192 (2007).

66. Galbraith, D. Flow cytometry and cell sorting: the next generation. Methods (San Diego, Calif 57, 249-250 (2012).

67. Chen, Y., et al. Review of advanced imaging techniques. Journal of pathology informatics 3, 22 (2012).68. Beum, P.V., Mack, D.A., Pawluczkowycz, A.W., Lindorfer, M.A. & Taylor, R.P. Binding of rituximab,

trastuzumab, cetuximab, or mAb T101 to cancer cells promotes trogocytosis mediated by THP-1 cells and monocytes. J Immunol 181, 8120-8132 (2008).

69. Beum, P.V., et al. Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells. J Immunol 187, 3438-3447 (2011).

70. Hudrisier, D., et al. Ligand binding but undetected functional response of FcR after their capture by T cells via trogocytosis. J Immunol 183, 6102-6113 (2009).

71. Williams, M.E., et al. Thrice-weekly low-dose rituximab decreases CD20 loss via shaving and promotes enhanced targeting in chronic lymphocytic leukemia. J Immunol 177, 7435-7443 (2006).

72. Lieschke, G.J. & Burgess, A.W. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (1). The New England journal of medicine 327, 28-35 (1992).

73. Lieschke, G.J. & Burgess, A.W. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (2). The New England journal of medicine 327, 99-106 (1992).

74. Amulic, B., Cazalet, C., Hayes, G.L., Metzler, K.D. & Zychlinsky, A. Neutrophil function: from mechanisms to disease. Annual review of immunology 30, 459-489 (2012).

75. Colombo, M.P., et al. Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo. The Journal of experimental medicine 173, 889-897 (1991).

76. Di Carlo, E., et al. The intriguing role of polymorphonuclear neutrophils in antitumor reactions. Blood 97, 339-345 (2001).

77. Midorikawa, Y., Yamashita, T. & Sendo, F. Modulation of the immune response to transplanted tumors in rats by selective depletion of neutrophils in vivo using a monoclonal antibody: abrogation of specific transplantation resistance to chemical carcinogen-induced syngeneic tumors by selective depletion of neutrophils in vivo. Cancer research 50, 6243-6247 (1990).

36

Chapter 1

78. Suttmann, H., et al. Neutrophil granulocytes are required for effective Bacillus Calmette-Guerin immunotherapy of bladder cancer and orchestrate local immune responses. Cancer research 66, 8250-8257 (2006).

79. van Egmond, M. Neutrophils in antibody-based immunotherapy of cancer. Expert opinion on biological therapy 8, 83-94 (2008).

80. Siders, W.M., et al. Involvement of neutrophils and natural killer cells in the anti-tumor activity of alemtuzumab in xenograft tumor models. Leukemia & lymphoma 51, 1293-1304 (2010).

81. Hernandez-Ilizaliturri, F.J., et al. Neutrophils contribute to the biological antitumor activity of rituximab in a non-Hodgkin’s lymphoma severe combined immunodeficiency mouse model. Clinical cancer research : an official journal of the American Association for Cancer Research 9, 5866-5873 (2003).

82. van der Kolk, L.E., Grillo-Lopez, A.J., Baars, J.W. & van Oers, M.H. Treatment of relapsed B-cell non-Hodgkin’s lymphoma with a combination of chimeric anti-CD20 monoclonal antibodies (rituximab) and G-CSF: final report on safety and efficacy. Leukemia 17, 1658-1664 (2003).

83. Niitsu, N., et al. Phase I study of Rituximab-CHOP regimen in combination with granulocyte colony-stimulating factor in patients with follicular lymphoma. Clinical cancer research : an official journal of the American Association for Cancer Research 10, 4077-4082 (2004).

84. Cartron, G., et al. Granulocyte-macrophage colony-stimulating factor potentiates rituximab in patients with relapsed follicular lymphoma: results of a phase II study. J Clin Oncol 26, 2725-2731 (2008).

85. Nimmerjahn, F. & Ravetch, J.V. Antibodies, Fc receptors and cancer. Current opinion in immunology 19, 239-245 (2007).

86. Ravetch, J.V. & Bolland, S. IgG Fc receptors. Annual review of immunology 19, 275-290 (2001).87. Valerius, T., et al. Activated neutrophils as effector cells for bispecific antibodies. Cancer Immunol

Immunother 45, 142-145 (1997).88. Stockmeyer, B., et al. Preclinical studies with Fc(gamma)R bispecific antibodies and granulocyte colony-

stimulating factor-primed neutrophils as effector cells against HER-2/neu overexpressing breast cancer. Cancer research 57, 696-701 (1997).

89. Schneider-Merck, T., et al. Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage. J Immunol 184, 512-520 (2009).

90. Valerius, T., et al. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy. Blood 82, 931-939 (1993).

91. van Spriel, A.B., van Ojik, H.H. & van De Winkel, J.G. Immunotherapeutic perspective for bispecific antibodies. Immunology today 21, 391-397 (2000).

92. Curnow, R.T. Clinical experience with CD64-directed immunotherapy. An overview. Cancer Immunol Immunother 45, 210-215 (1997).

93. Fury, M.G., Lipton, A., Smith, K.M., Winston, C.B. & Pfister, D.G. A phase-I trial of the epidermal growth factor receptor directed bispecific antibody MDX-447 without and with recombinant human granulocyte-colony stimulating factor in patients with advanced solid tumors. Cancer Immunol Immunother 57, 155-163 (2008).

94. Honeychurch, J., et al. Therapeutic efficacy of FcgammaRI/CD64-directed bispecific antibodies in B-cell lymphoma. Blood 96, 3544-3552 (2000).

95. Repp, R., et al. Phase I clinical trial of the bispecific antibody MDX-H210 (anti-FcgammaRI x anti-HER-2/neu) in combination with Filgrastim (G-CSF) for treatment of advanced breast cancer. British journal of cancer 89, 2234-2243 (2003).

96. van Ojik, H.H., et al. CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations. Cancer research 63, 5595-5600 (2003).

97. Bakema, J.E. & van Egmond, M. Immunoglobulin A: A next generation of therapeutic antibodies? mAbs 3, 352-361 (2011).

98. Deo, Y.M., Sundarapandiyan, K., Keler, T., Wallace, P.K. & Graziano, R.F. Bispecific molecules directed to the Fc receptor for IgA (Fc alpha RI, CD89) and tumor antigens efficiently promote cell-mediated cytotoxicity of tumor targets in whole blood. J Immunol 160, 1677-1686 (1998).

99. Lohse, S., et al. Recombinant dimeric IgA antibodies against the epidermal growth factor receptor mediate effective tumor cell killing. J Immunol 186, 3770-3778 (2011).

100. Valerius, T., et al. FcalphaRI (CD89) as a novel trigger molecule for bispecific antibody therapy. Blood 90, 4485-4492 (1997).

101. Otten, M.A., et al. FcR gamma-chain dependent signaling in immature neutrophils is mediated by FcalphaRI, but not by FcgammaRI. J Immunol 179, 2918-2924 (2007).

102. Otten, M.A., et al. Immature neutrophils mediate tumor cell killing via IgA but not IgG Fc receptors. J

37

Chapter 1

1Immunol 174, 5472-5480 (2005).

103. van der Steen, L., et al. Immunoglobulin A: Fc(alpha)RI interactions induce neutrophil migration through release of leukotriene B4. Gastroenterology 137, 2018-2029 e2011-2013 (2009).

104. Otten, M.A., et al. Enhanced FcalphaRI-mediated neutrophil migration towards tumour colonies in the presence of endothelial cells. European journal of immunology 42, 1815-1821 (2012).

105. Bakema, J.E. & van Egmond, M. The human immunoglobulin A Fc receptor FcalphaRI: a multifaceted regulator of mucosal immunity. Mucosal immunology 4, 612-624.

106. Launay, P., et al. Fcalpha receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger’s disease). Evidence for pathogenic soluble receptor-Iga complexes in patients and CD89 transgenic mice. The Journal of experimental medicine 191, 1999-2009 (2000).

107. van Egmond, M., et al. Human immunoglobulin A receptor (FcalphaRI, CD89) function in transgenic mice requires both FcR gamma chain and CR3 (CD11b/CD18). Blood 93, 4387-4394 (1999).

108. Beyer, T., et al. Serum-free production and purification of chimeric IgA antibodies. Journal of immunological methods 346, 26-37 (2009).

109. Duchez, S., et al. Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation. Proceedings of the National Academy of Sciences of the United States of America 107, 3064-3069 (2010).

110. Pascal, V., et al. Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells. Haematologica 97, 1686-1694 (2012).

111. Amulic, B., Cazalet, C., Hayes, G.L., Metzler, K.D. & Zychlinsky, A. Neutrophil function: from mechanisms to disease. Annual review of immunology 30, 459-489.

112. Lichtenstein, A. Mechanism of mammalian cell lysis mediated by peptide defensins. Evidence for an initial alteration of the plasma membrane. The Journal of clinical investigation 88, 93-100 (1991).

113. Horner, H., et al. Intimate cell conjugate formation and exchange of membrane lipids precede apoptosis induction in target cells during antibody-dependent, granulocyte-mediated cytotoxicity. J Immunol 179, 337-345 (2007).

114. Kushner, B.H. & Cheung, N.K. Clinically effective monoclonal antibody 3F8 mediates nonoxidative lysis of human neuroectodermal tumor cells by polymorphonuclear leukocytes. Cancer research 51, 4865-4870 (1991).

115. Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science (New York, N.Y 303, 1532-1535 (2004).

116. Saffarzadeh, M., et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PloS one 7, e32366 (2012).

117. van Spriel, A.B., van Ojik, H.H., Bakker, A., Jansen, M.J. & van de Winkel, J.G. Mac-1 (CD11b/CD18) is crucial for effective Fc receptor-mediated immunity to melanoma. Blood 101, 253-258 (2003).

118. Zhou, M.J. & Brown, E.J. CR3 (Mac-1, alpha M beta 2, CD11b/CD18) and Fc gamma RIII cooperate in generation of a neutrophil respiratory burst: requirement for Fc gamma RIII and tyrosine phosphorylation. The Journal of cell biology 125, 1407-1416 (1994).

119. Grossman, W.J. & Ley, T.J. Granzymes A and B are not expressed in human neutrophils. Blood 104, 906-907; author reply 907-908 (2004).

120. Hochegger, K., Eller, P. & Rosenkranz, A.R. Granzyme A: an additional weapon of human polymorphonuclear neutrophils (PMNs) in innate immunity? Blood 103, 1176 (2004).

121. Metkar, S.S. & Froelich, C.J. Human neutrophils lack granzyme A, granzyme B, and perforin. Blood 104, 905-906; author reply 907-908 (2004).

122. Wagner, C., et al. Granzyme B and perforin: constitutive expression in human polymorphonuclear neutrophils. Blood 103, 1099-1104 (2004).

123. Stockmeyer, B., et al. Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells. J Immunol 171, 5124-5129 (2003).

124. Bakema, J.E., et al. Targeting FcalphaRI on polymorphonuclear cells induces tumor cell killing through autophagy. J Immunol 187, 726-732 (2011).

125. Mizushima, N., Levine, B., Cuervo, A.M. & Klionsky, D.J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069-1075 (2008).

126. Shen, S., Kepp, O. & Kroemer, G. The end of autophagic cell death? Autophagy 8, 1-3 (2012).127. Mantovani, A., Cassatella, M.A., Costantini, C. & Jaillon, S. Neutrophils in the activation and regulation of

innate and adaptive immunity. Nat Rev Immunol 11, 519-531 (2011).128. Pelletier, M., et al. Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 115, 335-

343 (2010).129. Fridlender, Z.G., et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: “N1” versus

38

Chapter 1

“N2” TAN. Cancer cell 16, 183-194 (2009).130. Costantini, C. & Cassatella, M.A. The defensive alliance between neutrophils and NK cells as a novel arm

of innate immunity. Journal of leukocyte biology 89, 221-233 (2011).131. Jaeger, B.N., et al. Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis.

The Journal of experimental medicine 209, 565-580 (2012).132. van Gisbergen, K.P., Geijtenbeek, T.B. & van Kooyk, Y. Close encounters of neutrophils and DCs. Trends

in immunology 26, 626-631 (2005).133. van Gisbergen, K.P., Sanchez-Hernandez, M., Geijtenbeek, T.B. & van Kooyk, Y. Neutrophils mediate

immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN. The Journal of experimental medicine 201, 1281-1292 (2005).

134. Bennouna, S., Bliss, S.K., Curiel, T.J. & Denkers, E.Y. Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. J Immunol 171, 6052-6058 (2003).

135. Megiovanni, A.M., et al. Polymorphonuclear neutrophils deliver activation signals and antigenic molecules to dendritic cells: a new link between leukocytes upstream of T lymphocytes. Journal of leukocyte biology 79, 977-988 (2006).

136. Gelderman, K.A., Tomlinson, S., Ross, G.D. & Gorter, A. Complement function in mAb-mediated cancer immunotherapy. Trends in immunology 25, 158-164 (2004).